1a-phthalylamino acid derivatives of mitomycin c



Fish. 3, 1970" MASANAO MATSUI er'AL. 3,493583' la-PHTHALYLAMINO ACID DERIVATIVES OF MITOHYGIN C Filed Nov. 28, 1967 4 Sheets-Sheet 1 700E" ran:

Ayn-m0 name, "an! M4001 l. 770/! MIT-S Feb. 3, 1970 msmm mm m. 3,493,583

le-PHTHALYLAMINO ACID DERIVATIVES 0F MITOIYCIN 0 Filed Nov. 28, 1967 4 Sheets-Sheet 2 arr Ida/J64- Feb. 3, 1970 MASANAO MATSUI AL IQ-PHTHALYLAMINO ACID DERIVATIVES 0F MITOMYCIN C Filed NOV. 28, 1967 FIG. 7

4 Sheets-Sheet 4 M/l/EM r025 #14 .m #44 nan-w, may mm nara r NAKAIt/ rosy rpg Sosa! A rranven United States Patent ABSTRACT OF THE DISCLOSURE Compounds of the formula:

0 onto (3 ONE:

wherein R is a member selected from the group consisting of /CH2, OH; C 115 /CH, CH;

H, CH3,

The present application is a continuation-in-part of copending application Ser. No. 482,382, filed on Aug. 25, 1965, and now abandoned.

The present invention relates to novel derivatives of mitomycin C and, more particularly, to la-phthalylamino acid derivatives of mitomycin C.

In 1956, new antibiotics termed mitomycin A and mitomycin B having, inter alia, intense antibacterial properties were found in the culture medium of Streptomyces caespitosus. Subsequently, mitomycin C was isolated from a culture medium of the same strain. However, the clinical applications of mitomycin C are limited because of its strong toxicity.

Recently, the chemical structure of mitomycin C was clarified as being as follows:

CH2O C ONH2 3,493,583 Patented Feb. 3, 1970 rice invention are those represented by the following Formula II:

0 c1120 0 ONE! l I 0 CH3 HSN O H 00 II I No I N 0 0 (II) wherein R is a residue from an amino acid, excluding the 1\|IH2 1c 0 OH group.

As is already known in the art, phthalylized amino acids can be obtained by adding amino acids to phthalic anhydride followed by heating. Furthermore, in order to elevate the reactivity thereof, a halogenating agent such as a phosphorous halide or thionyl chloride is reacted in order to obtain a phthalyl amino acid halide, shown by Formula III:

R N COX H \OO (III) wherein R is the same as shown in Formula II hereinabove and X is halogen. When the above-described phthalyl amino acid halides and mitomycin C are allowed to stand in the presence of a base, novel derivatives can Various kinds of compounds can be used as the base, but oxides of alkaline earth metals such as magnesium oxide or organic bases such as triethylamine give a particularly favorable result.

The novel derivatives, obtained in accordance with the present invention as shown hereinabove, are diiferent from mitomycin C with respect to their infra-red absorption spectra, their ultraviolet absorption spectra as well as in the analyzed values obtained therewith. For example, they showobviously different behaviors on a column chromatogram and a thin layer chromatogram for which silica gel is used.

In the attached drawings:

FIGURE 1 shows an ultraviolet absorption curve of 1aphthalyl alanyl mitomycin C,

FIGURE 2 shows an infra-red absorption curve of 1aphthalyl alanyl mitomycin C,

FIGURE 3 illustrates an ultraviolet absorption curve of la-phthalyl phenylalanyl mitomycin C,

FIGURE 4 shows an infra-red absorption curve of 1aphthalyl phenylalanyl mitomycin C,

FIGURE 5 shows an ultraviolet absorption curve of la-phthalyl leucyl mitomycin C,

FIGURE 6 illustrates an infra-red absorption curve of la-phthalyl leucyl mitomycin C,

FIGURE 7 shows an infrared absorption curve of 1aphthalyl glycyl mitomycin C,

FIGURE 8 is an infra-red absorption curve of 1aphthalyl valyl mitomycin, and

FIGURE 9 illustrates an infra-red absorption curve of la-phthalyl isoleucyl mitomycin C.

The toxicities of the novel mitomycin derivative obtained by the present invention are shown in Table 1:

TABLE 1 Derivatives: LD mg./ kg. (mouse) la-phthalyl-alanyl mitocycin C 100 la-phthalyl-phenylalanyl mitomycin C 100 la-phthalyl-leucyl mitomycin C 100 la-phthalyl-isoleucyl mitomycin C 200 la-phthalyl-valyl mitomycin C 200 On the basis of these in vivo tests in mice, it can be seen that the novel derivatives of the present invention are less toxic than mitomycin C itself.

The new compounds of the present application are useful as antibacterial agents and have broad-spectrum antibacterial activity. The antibacterial spectrum of typi cal compounds of this invention, representing the amount required to inhibit the growth of various typical bacteria, was determined in a standard manner by the agar plate culture method. The minimum inhibition concentrations of these derivatives against various kinds of bacilli, expressed in micrograms per millilter (mcg./ml.), are shown in Table 2.

with gentle shaking. This reaction mixture Was concentrated under reduced pressure to obtain 4.7 g. of viscous phthalyl-alanyl chloride.

Subsequently, 668 mg. of mitomycin C were dissolved in 30 ml. of dry dioxane, 80 mg. of magnesium oxide powder were added, and then 522 mg. of phthalyl-alanyl chloride obtained by the above process were dissolved in 15 ml. of dry dioxane and further added thereto. This mixture was allowed to stand for hours at room temperature after 2 hours of shaking at room temperature.

The reaction solution was subjected to the silica gelchloroform system chromatogram and developed with dioxane. The dioxane effiuent was solidified by addition of ligroin after evaporating the solvent on a water bath kept at 30 C., washed with ether and the resulting powder was separated and dried.

Recovery: 80%

Molecular formula:

Analyzed values: C, 58.66%; H, 5.50%; N, 11.56%

Theoretical values: C, 59.88%; H, 4.82%; N, 10.74%

C2 H25O N5. Molecular Weight! Example II Micrograms per milliliter laphthalyl la-phthalylla-phthalylla-phthalyb la-phthalylalanyl phenylalanyl leucyl lsoleucyl valyl Bacilli examined mitomycin C mitomycin C mitomycin C mitomycin C mitomycin C Staphylococcus aareus 2091 25 6. 25 3. 12 50 Sarcina latea Pcl 1001 25 6. 25 12.5 50 50 Bacillus subtilis A'ICC 6633 25 50 6. 25 50 50 Salmonella typhi 379 50 50 50 50 50 Shigella flczneri Za 3196 1. 56 3. 12 25 50 50 K lebstella pneumoniae 0/10 50 50 50 50 50 Proteus X19 50 50 50 50 50 Escherichia cali K-l2 50 50 25 50 50 Pseudomonas aeruginosa 3 50 50 50 50 50 Vtbrio comma 62 3. 12 1. 56 6. 25 50 50 Mycobacterium tuberculosis 607. O. 78 0. 78 O. 78 50 50 Streptococcus hacmolyticus 68 6. 25 1. 56 6. 25 50 50 Streptococcus faecalis 5 25. 12. 5 25 50 50 Co'rgmcbacterlum diphtheriae 92 3. 12 3. 12 3. 12 50 1 252 Diplococcus penu-mimiae I 19 The following examples are given merely as illustrative of the present invention and are not to be considered as limiting. Unless otherwise noted, the percentages therein and throughout the application are by weight.

Example I 4.5 g. of L-alanine and 7.4 g. of phthalic anhydride were mixed and charged into a 300 ml. flask equipped with a reflux means and a water separator, and 150 ml. of toluene and 2 ml. of triethylamine were added. This was followed by heating at 155165 C. on an oil bath for 2 hours. Twenty ml. of water were added to a residue obtained by concentrating the above reaction mixture under reduced pressure. The crystals deposited were filtered and dissolved in ethyl alcohol, followed by filtration. The filtrate was concentrated, 16 ml. of water and 4 ml. of concentrated hydrochloric acid were added, and the resultant deposited white crystals were washed and filtered to obtain phthalyl-alanine. 4.3 g. of the phthalyl-alanine were added to 4.1 g. of phosphorus pentachloride and 30 ml. of dried benzene charged in a 100 ml. flask to which a calcium chloride tube was attac ed a d k pt t 60 C. on a W te ba f r 2 hou s as by filtration. As a result, 12.4 g. of phthalyl phenylalanine were obtained.

Subsequently, 9.0 g. of phosphorus pentachloride and ml. of dry benzene were charged into a flask of 200 ml. capacity, to which 12.7 g. of N-phthalyl-L-phenylalanine were added, and this reaction mixture was allowed to stand for 2 hours at 60 C. on a water bath with gentle shaking with the aid of an attached calcium chloride tube.

1.33 g. of phthalyl-phenylalanyl chloride were obtained pressure.

603 mg. of mitomycin C were dissolved in 30 ml. of dry dioxane, followed by the addition of 72 mg. of magnesium oxide powder as well as by the further addition of a solution prepared by dissolving 603 mg. of phthalylphenylalanyl chloride obtained above in 15 ml. of dry dioxane. This mixture was allowed to stand for 37 hours at room temperature after 2 hours of shaking at room temperature. The reaction solution was subjected to the silica gel-chloroform system chromatogram and developed with dioxane. The dioxane efiluent was taken out and, after evaporating the solvent on a water bath at 30 (2., treated with ligroin and then with ether to 0b-= tain a powder. The reaction product was obtained after drying.

Recovery: 60%

Molecular formula:

Analyzed values: C, 62.44%; H, 5.47%; N, 10.92%

Theoretical values: C, 62.84%; H, 4.78%; N, 11.45%

Example III 6.6 g. of L-leucine and 7.4 g. of phthalic anhydride were mixed in a flask of 300 ml. capacity with the further addition of 150 ml. of toluene and 2 ml. of triethylamine and heated for 2 hours on an oil bath at 155165 C.

Two ml. of concentrated hydrochloric acid and 20 ml. water were added to a residue obtained by concentrating the reaction mixture under reduced pressure and the deposited white crystals were filtered to obtain phthalyl- Ieucine.

5.6 g. of phosphorus pentachloride and 100 ml. of dry benzene were successively charged into a flash of 200 ml. capacity, to which 7.0 g. of phythalyl-leucine, obtained above, was added. This mixture was allowed to stand for 2 hours at 60 C. with gentle shaking. 560 mg. of the reaction mixture concentrated under reduced pressure were dissolved in ml. of dioxane, 668.6 mg. of mitomycin C were dissolved in 30 ml. of dry dioxane, to which 80 mg. of magnesium oxide were added, and after admixing the two solutions, they were allowed to stand for 23 hours at room temperature after shaking for 2 hours at room temperature. The reaction solution was subjected to the silica gel-chloroform system chromatogram and developed with dioxane. The dioxane efiluent was taken out and, after evaporating the solvent on a water bath at 30 C., subjected to a powdering treatment with ligroin followed by ether and finally dried.

C32H290 N5. Molecular Weight:

Recovery: 83%

Molecular formula: C H O N Molecular weight: 577.6 Analyzed values: C, 59.58% H, 5.93% N, 10.38% Theoretical values: C, 61.20% H, 5.32% N, 10.20%

Example IV 3.9 g. of glycine and 7.4 g. of phthalic anhydride were mixed in an egg plant-shaped flask of 100 ml. capacity, kept at 170180 C. on an oil bath for 30 minutes and allowed to stand at room temperature, followed by recrystallization from ethanol. As a result, 5.9 g. of phthalylglycine were obtained.

3.1 g. of phthalyl-glycine, obtained in a 100 ml. flask as mentioned above, and 12 ml. of thionyl chloride were subsequently reached at 85 C. for 1 hour, followed by recrystallization from benzene-petroleum ether to obtain 24 g. of phthalyl chloride.

1,003 mg. of mitomycin C were dissolved in 40 ml. of dry dioxane to which 100 mg. of magnesium oxide powder were added successively with the addition of a solution prepared by dissolving 737 mg. of the phthalyl-glycyl chloride obtained above in 10 ml. of dry dioxane, and the mixture was allowed to stand for 37 hours at room temperature after 2 hours of shaking at room temperature. The reaction solution was subjected to the silica gel-chloroform system chromatogram, developed with dioxane, the dioxane effluent was concentrated at 30 C. on a water bath, powdered with ligroin followed by ether and further dried to obtain the product derivative.

Yield: 1250 mg. Recovery: 80%

Molecular formula: C H O N Molecular Weight: 521.5 Analyzed values: C, 56.98%; H, 5.3%; N, 12.64% Theoretical values: C, 57.58%; H, 4.44%; N, 13.43%

Example V The process was carried out according to that of Example IV using 1.0 g. of L-valine and 1.26 g. of phthalic anhydride to obtain 1.7 g. of phthalyl-valine.

Subsequently 1.0 g. of phthalyl-valine and thionyl chloride were treated by the process described in Example IV to obtain 0.8 g. of phthalyl-valyl chloride.

1,003 mg. of mitromycin C and 877 mg. of phthalylvalyl chloride were treated similarly as in Example IV and 1,705 mg. of the product were obtained.

Recovery: 95.4%

Molecular formula: C H O N Molecular weight: 578.6 Analyzed values: C, 58.02%; H, 6.10%; N, 11.98% Theoretical values: C, 59.56%; H, 5.35%; N, 12.40%

Example V1 Recovery: 90.0%

Molecular formula: C H 0 N Molecular weight: 57 8.6 Analyzed values: C, 58.99%; H, 5.20%; N, 12.30% Theoretical valuesz C, 60.20%; H, 5.57%; N, 12.11%

Example VII 2.8 g. of L-methionine and 2.8 g. of phthalic anhydride were used and treated according to the process described in Example IV to obtain 5.1 g. of phthalyl-methionine.

Subsequently, 3.2 g. of phthalyl-methionine and thionyl chloride were treated by the process of Example IV and, as a result, 2.5 g. of phthalyl-methionyl chloride were obtained.

Using 1,003 mg. of mitomycin C and 979 mg. of phthalyl-methionyl chloride, the process was carried out similarly to that described in Example IV to obtain 1,564 mg. of product.

Recovery: 87.5%

Molecular formula: C H O N S. Molecular weight:

Analyzed values: C, 55.54%; H, 4.80%; N, 12.10% Theoretical values: C, 56.45%; H, 4.91% N, 11.76%

We claim: 1. A compound of the formula:

wherein R is a member selected from the group consisting of C H: C H: C H3 CH3, CH, CHI-CH2, CH and CH2 CH3 CH3 C2H5 2. 1a-phthalyl-alanyl mitomycin C.

3. la-phthalyl-phenylalanyl mitomycin C. 4. la-phthalyl-leucyl mitomycin C.

5. la-phthalyl-valyl mitomycin C.

6. la-phthalyl-isoleucyl mitomycin C.

7 7. la-phthalyl-glycyl mitomycin C. 8. la-phthalyl-methionyl mitomycin C.

References Cited UNITED STATES PATENTS 8 ALEX MAZEL, Primary Examiner J. A. NARCAVAGE, Assistant Examiner US. Cl. X.R. 5 424-274 3,226,393 12/1965 Meyer 260-295 

